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The association between Mi-2 antibodies and dermatomyositis.

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Antibodies to Mi, an antigen in calf thymus
extract, have been demonstrated by complement fixation inhibition in polymyositis (PM) and dermatomyositis (DM) sera but not in the sera of individuals without
myositis. The original Mi reference serum defined 2
precipitating antibodies, using immunodiffusion (ID).
Anti-Mi-1 was not active in complement fixation. We
have now studied in further detail anti-Mi-2, which
appears to be the antibody in Mi serum that fixes
complement. Mi-2 antigen was purified by immunoaffinity chromotography. An enzyme-linked immunosorbent assay (ELISA) to measure Mi-2 antibody, using
this antigen, was used to test the sera of 139 myositis
patients: 52 had DM and 87 had PM. Control sera from
35 normal subjects and 93 patients with other connective
tissue diseases were also tested. Only 13 sera were
considered definitely positive for anti-Mi-2. All were
from patients who had myositis, 11 of whom had DM.
Only DM sera had anti-Mi3 by ID, and all sera with
anti-Mi3 by ID were positive by ELISA. A number of
other sera, including many from patients with other
connective tissue diseases and 2 from normal subjects
From the Veterans Administration Medical Center, the
University of Oklahoma Health Sciences Center, and the Arthritis
and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma.
Supported by VA Medical Research funds from the Veterans Administration Medical Center and by NIH grant AM-32214.
Ira N. Targoff, MD: Associate Investigator, Veterans Administration; Morris Reichlin, MD: Professor of Medicine, Chief,
Combined Immunology Section, Department of Medicine, University of Oklahoma Health Sciences Center, and Head, Arthritis and
Immunology Program, Oklahoma Medical Research Foundation.
Address reprint requests to Ira N. Targoff, MD, Arthritis
and Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104.
Submitted for publication October 16, 1984; accepted in
revised form January 30, 1985.
Arthritis and Rheumatism, Vol. 28, No. 7 (July 1985)
(all without precipitating antibodies) had lower elevations which were of uncertain significance. Detection of
anti-Mi-2 by ID as well as by ELISA was significantly
more frequent in DM than in PM. Anti-Mi3 appears to
be closely linked to DM, and is the first specific serologic
marker for this form of myositis.
Autoantibodies can be found in the serum of
almost 90% of patients who have polymyositis (PM) or
dermatomyositis (DM) (1). The sera of 60% of these
patients form precipitates with saline tissue extracts
on Ouchterlony immunodiffusion (ID), and the others
stain tissue culture substrates, such as HEp-2 cells, by
indirect immunofluorescence (IIF). The antibodies involved in the precipitates in ID can be specifically
identified by comparison with reference sera for each
described specificity.
At least 8 precipitating autoantibody specificities have been identified in myositis patients ( 2 ) , and
there are many more that are as yet uncharacterized.
Each individual antibody specificity occurs in only a
fraction of the patients; the most common, anti-Jo-l ,
is seen in fewer than 20% (3). Most of these antibodies
are limited to, or found with increased frequency in,
patients who can be classified into characteristic clinical subgroups of the disease. Almost all patients with
antibodies to Jo-1, PM-Scl (PM-I), and Ku (4) have
myositis or overlap syndromes including myositis.
Nuclear RNP (nRNP), Sm, Ro (SS-A), and La (SS-B)
antibodies are usually seen in patients who have
overlap syndromes with myositis, and are more frequently associated with other connective tissue diseases.
The Mi specificity was originally described in
Mi serum by a complement fixation (CF) reaction and
was defined in other sera by the ability of their papain
digests to specifically inhibit this reaction (5). It appeareld to be specific for myositis and was found in
60% of PM and DM sera. T h e reference serum for this
reaction formed 2 precipitin lines on ID, Mi-I and Mi2. Mi-l antigen has been described in detail (6). Since
anti-Mi-I does not fix complement, it was clearly not
the original Mi antibody, and furthermore, its occurrence was not limited to myositis patients (7). Mi-2
antigen (Mi-2 Ag), on the other hand, has not been
T h e current investigation was an effort t o learn
more about the Mi antibody responsible for the original CF reaction that appeared to be so common in
myositis. It appears increasingly likely that this is the
Mi-2 antibody recognized by ID. Anti-Mi-2 is present
at a lower frequency than the CF antibody, but this
study reveals a higher rate of the precipitin than
previously recognized. We have studied the Mi-2 Ag in
more detail and have developed a sensitive enzymelinked immunosorbent assay (ELISA) t o measure
anti-Mi-2. This has been used t o survey a large
population of myositis patients, normal subjects, and
patients with other connective tissue diseases. An
association between anti-Mi-2 and DM has been
. .
. .
N 4 7
Sera. Patient Mi is a 60-year-old woman with chronic, classic DM of >I0 years duration, with continuing
evidence of disease and a persistently high titer of Mi
antibody. For most of the studies reported here, her plasma
was collected in 1981 by plasmapheresis and stored at
-20°C. This sample had an anti-Mi-2 precipitin titer of 1:32
using 'calf thymus extract as antigen. Other sera used in this
study have been collected over a period of 10 years and
stored at -20°C. Serum samples from 139 patients with a
diagnosis of PM or DM, divided into 4 groups as indicated in
Figure I , were tested. Fifty-one patients were seen in
Buffalo or Oklahoma City, 68 in Baltimore, and 20 in other
Serum was included in this study if the patient had
clinical proximal muscle weakness, an elevated level of
muscl'e enzymes, and evidence of myositis shown by electromyography, muscle biopsy, or both, as in the criteria of
Bohari and Peter (8). Those with a characteristic rash were
classified as having DM (childhood or adult). Two adult
patients with DM had overlap syndrome, and at least 7 DM
patients had an associated malignancy. PM patients (those
without a rash) were categorized according to those who had
PM alone and those who had overlap with another connective tissue disease, either systemic lupus erythematosus
(SLE), progressive systemic sclerosis (PSS), rheumatoid
arthritis (RA), or Sjogren's syndrome. In addition, serum
sampl'es from 35 normal adults, the majority of whom were
Figure 1. Anti-Mi-2 activity of 267 sera as measured by enzymelinked immunosorbent assay. Conversion of optical density to units
was done by comparison with a standard curve of Mi serum as
described in Patients and Methods. All precipitin-positive sera fall
on or above the upper horizontal line. All positive sera were from
patients with myositis, most of whom had dermatomyositis (DM).
PM = polymyositis; cDM = childhood DM; PMol = PM overlap;
N L = normal; SLE = systemic lupus erythematosua: RA =
rheumatoid arthritis; PSS = progressive systemic sclerosis.
young adult laboratory personnel, and from 93 patients with
other connective tissue diseases (SLE, RA, or PSS) were
tested as controls.
Antigen purification. Calf thymus extract (CTE) was
used as the source of antigen for most procedures. It was
obtained fresh and either stored frozen between -40°C and
-70°C or used immediately. Extract was prepared as described previously (9). After ammonium sulfate fractionation, the extract was either dialyzed to 0.05M Tris-HCI (pH
7.2), 0.5M NaCI, and 0.01M sodium azide (TBS) for affinity
chromatography or was equilibrated with 0.01M phosphate
buffer at pH 7.0 and passed through a similarly equilibrated
DEAE-cellulose column. After washing, this column was
eluted with stepwise increases in NaCl concentration. Antigen was concentrated to approximately 50 mg of proteinhl
and applied to a 300-ml Sepharose 6B column. Antigenic
activity was detected by ability to fix complement with Mi
serum, or by ID, and later by ELISA.
The IgG fraction of 30 ml of Mi serum was prepared
according to the method of Jaton et al (lo), yielding 10.5 mg/
ml. This was coupled, according to manufacturer's instructions, to cyanogen bromide-preactivated Sepharose 4B
(Pharmacia, Piscataway, NJ) at 5 mg per ml of gel, resulting
in >98% coupling. The gel was then washed with alternating
pH buffers, then with 4M MgC12, then equilibrated with
TBS, and finally, packed in a column. This immunoadsorbent was used to purify Mi-2 Ag for most of the experiments
described. Extract o r partially purified material was applied
to the column, and the column was washed with TBS and
eluted with 4M MgClz at p H 7.0. Protein-containing eluate
was dialyzed back to 0.5M NaCI, concentrated, then dialyzed to 0.01M phosphate buffer, pH 7.2, 0.15M NaCI, and
0.01M sodium azide (PBS), and stored at -70°C.
For final purification, it was necessary to remove
contaminating bovine gamma globulin (BGG). Pre-purification steps did not remove all BGG, nor did affinity chromatography, since Mi reference serum also contained anti-Mi1, which is an antibody that has been shown to bind BGG (6,
7). BGG therefore bound to the affinity column and was
eluted along with Mi-2. BGG (and Mi-1, also bound by antiBGG) was removed by a n anti-BGG immunoadsorbent.
Purified specific rabbit anti-BGG was prepared from rabbit
antiserum to BGG with a column of BGG (Cohn fraction 11)
coupled to Sepharose 4B. The 1M acetic acid eluate of this
column was dialyzed back to bicarbonate buffer at pH 8.3
and in turn coupled to Sepharose 4B. This gel had a BGG
adsorption capacity of 6 mg per ml of gel. A 5.2-ml column
was used (usually for <2 mg total protein) and regenerated
prior to each run.
ELISA for anti-Mi-2. In each of the 4 steps of the
ELISA, 0.1 ml volume was added, and the plates were
incubated overnight at 4"C, then washed 5 times with PBS
containing 0.05% Tween 20. Ninety-six-well Dynatech Immulon I plates were coated with purified Mi-2 Ag at 3 pg/rnl
for most preparations. Serum was usually screened at a
1:lOO dilution in a blocking solution consisting of PBS with
0.05% Tween 20, 10 mg/ml bovine serum albumin (Cohn
fraction V), and 1 mg/ml BGG. The latter was added to block
any reaction, by antibodies to BGG o r Mi-1, with traces of
BGG which might still be present in the antigen preparation.
On plates coated directly with 10 pg/ml BGG, the anti-BGG
activity of Mi serum by ELISA was blocked by I mgiml
BGG. A 1:l ,OOO dilution of affinity-purified goat anti-human
IgG (gamma chain-specific) conjugated to alkaline phosphatase (Sigma, St. Louis, MO) was then added. For the final
step, the substrate, p-nitrophenyl phosphate, was added.
Plates were read by a Dynatech ELISA Reader at 405 nm
and 490 nm, usually at 15-minute intervals until I hour, or
until Mi serum at 1 : 100 dilution (which gave maximal color
development) reached an O D of > 1.
Each test was run in duplicate and the values were
averaged. Mi serum, at increasing tenfold dilutions from
]:lo0 to 1:100,000, was included on each plate. Mi serum
activity of 1:100,000 dilution was defined as I ELISA
activity unit. The calculated number of units in each dilution
of Mi serum tested was plotted against the corresponding
O D at that dilution, and the O D of each sample was used to
determine the number of units from this standard curve for
that run.
Table 1. Purification of Mi-2
100 gm tissue
Calf thymus extract
Ammonium sulfate
Ion exchange
Gel filtration
( P fixed)
40 I
3 1,688
For some experiments, the ability of antigen to
inhibit anti-Mi-2 was tested. When measuring the amount of
antigen activity, the preparation was added at the serum step
to inhibit Mi serum at 15,000 dilution (20 units). In order to
provide supporting evidence for o r against the presence of
specific antibody in some of the sera with low levels of
activity, the ability of antigen to inhibit this activity was
determined using as antigen either the Mi-2 Ag, original
CTE, o r CTE which had Mi-2 Ag removed by affinity
chromatography. Between 5 and 10 times the amount of
antigen required to inhibit 20 units of Mi serum by 50% of the
original OD was used (in most cases, 1 mg/ml of extract
preparations was used).
Other methods. Methods for performance of RNase,
DNase, and trypsin digestion (9), I D (7), and C F (9) have
been previously described. Polyacrylamide gel electrophoresis (PAGE) was performed by a previously described modification of the Laemmli method (6), and Western blotting was
done as described by Towbin et al(l1). Rat liver nuclei were
prepared by the method of Widnell and Tata (12), and the
initial supernatant was used as the cytoplasmic fraction.
Protein was measured by the Bio-Rad protein assay (13).
Antigen detection. The Mi-2 Ag could be detected by ID in both the 0-30% and the 30-60% ammonium sulfate fractions, with more total antigen from the
30-60% fraction which was used for most of the
experiments. Those fractions were also active by C F
with Mi serum. No antigen could be detected in the
flow-through fraction of DEAE chromatography by
either ID or CF. With NaCl elution, all antigen was
found between 0.1M and 0.2M NaCl. The antigen from
DEAE-purified extract behaved as a protein of
70,000-90,000 MW on a Sepharose 6B column. As
determined by CF, very modest purification could be
accomplished by these methods with substantial loss
of antigenic activity, while affinity chromatography
accomplished much greater purification, as shown in
Table 1. Eluting with acid or base destroyed C F
activity, but some activity could be retained with
was 329-fold purified. After adsorption twice by the
anti-BGG column, no BGG could be detected by ID.
Antigen characterization. Using sodium dodecyl
sulfate (SDS)-PAGE, the preparation showed 2 bands
and no protein impurities by Coomassie blue staining
when 5 pg of Mi-2 Ag was loaded on the gel, but faint
additional bands of 25,000-30,000 MW were seen with
silver stain. The molecular weights of the major bands
on a typical run were 53,000 and 61,000 daltons. When
Western blotting was performed, although transfer
was accomplished (according to protein-stained paper
Figure 2. Inibitiotl of the anti-Mi-2 activity of Mi serum (1:5,000
dilution). Treating the purified antigen with RNase did not affect
antigenic activity, as measured by ability to inhibit activity on
enzyme-linked immunosorbent assay. Inhibitory activity for treated
and untreated antigen was less than that shown in Figure 3, possibly
because of incubation for digestion.
MgC& elution. Affinity-purified antigen had to be concentrated considerably before ID activity could be
detected at 0.24 mg/ml. Attempts to further purify the
antigen have not yet been successful. CF activity was
purified 160-170-fold. As detected by ability to inhibit
the anti-Mi-2 ELISA by 50%, this Mi-2 preparation
1 .o
e-. R A T
1 o2
Figure 3. Inhibition of anti-Mi-2 by cell fraction extracts. Nuclear
extract was much more efficient at inhibiting activity on enzymelinked immunosorbent assay than was cytoplasmic extract. Rat liver
nuclear extract was an effective inhibitor of activity against calf
thymuisderived Mi-2 antigen coating the plate.
Figure 4. Mi-2 enzyme-linked immunosorbent assay, as described
in Patients and Methods. Typical dilution curve. Mi serum gave
much greater optical density compared with the others, reflecting
the amount of Mi antibody. Patient Go had dermatomyositis;
patients Wa and Ba had polymyositis.
and original gel), no increased staining could be detected on either band after addition of Mi serum or a
second anti-Mi-2-containing serum, compared with
normal serum or no serum. It appeared that antigenic
activity was destroyed by SDS-PAGE. After phenol
extraction of purified antigen and PAGE on urea gel,
no bands could be detected by ethidium bromide
Antigen that had been digested by trypsin lost
its ability to inhibit the anti-Mi-2 ELISA or form a
precipitin line in ID with Mi serum. However, RNase
digestion (which destroyed nRNP activity) did not
affect Mi-2 Ag activity by ELISA or ID (Figure 2). The
antigen was also resistant to DNase digestion.
IIF with Mi serum on HEp-2 cells or mouse
kidney substrate showed a homogeneous nuclear pattern with a titer >1:1,000. No cytoplasmic staining
was seen. Four other patients with Mi-2 precipitins
also had titers >l:l,OOO on IIF, with a homogeneous
pattern. Rat liver nuclear extract was very efficient at
inhibiting the anti-Mi-2 ELISA, while no inhibition
was seen with the cytoplasmic fraction at comparable
concentrations (Figure 3). These data support the
nuclear origin of the antigen.
Antibody to Mi-2 on ELISA. Mi-2 antigen was
quite efficient at coating wells for ELISA, reaching
saturation at 3 p g h l at both pH 9 and pH 7 . 2 . Activity
of Mi serum could be detected at
dilution, but
usually not at
A typical dilution curve is shown
in Figure 4, and typical antigen inhibition curves are
seen in Figure 5 . Some background was consistently
seen with normal serum at 1:100 dilution.
All the sera were screened at 1:lOO dilution and
Figure 5. Antigen inhibition of enzyme-linked immunosorbent assay (ELISA) activity. Purified Mi or calf thymus extract (CTE) was
added to Mi serum at a 1:5,000 dilution before adding to the plate.
Increasing antigen concentration decreased ELISA activity. Fifty
percent inhibition was achieved with purified antigen that was 329fold less concentrated than CTE.
all the results converted to ELISA units and plotted on
a scattergram, as seen in Figure 1 . A number of DM
patients had activity markedly above that seen in other
groups. Only myositis patients had 2400 units of
activity (defined as positive), and of these, 11 had DM
(1 child) and 2 had PM (without overlap). All of the IDpositive patients had 2400 units, and all had DM. One
of the PM samples was unusual, having fairly high
activity at 2,510 units but no precipitin and a low titer
(I :40) by IIF.
Two of the patients with anti-Mi-2 were known
to have malignancies (breast, thymoma) that appeared
to be associated with the DM. Another patient, who
appeared to have true antibody by inhibition studies
(see below), also had a breast malignancy. One patient
with anti-Mi-2 had overlap with SLE; this was the
only patient with anti-Mi-2 who had any other recognized autoantibody, anti-nRNP. Among those for
whom a detailed clinical history was known, there
were no distinctive unifying features.
All samples were first examined by routine
screening by ID with unpurified but concentrated CTE
(50-100 mg protein/ml). Anti-Mi-2 was detected and
was strong enough to be identified only in Mi serum
and 2 other sera. In retrospective testing of ELISApositive sera using a highly concentrated extract purified by DEAE chromatography, anti-Mi-2 could be
detected by ID in 7 of 9 DM sera tested (the 2 negative
sera having only 500 and 560 ELISA units) and 0 of 2
PM sera. Sera with low-level elevations of ELISA
activity (below 400 units) were still negative.
When the values for the normal subjects were
averaged and the number of units representing 2
standard deviations above the mean was determined
(140 units), it was found that 21 myositis patients (8
PM, 6 DM, and 7 PM overlap) and 17 controls (2
normal, 10 SLE, 3 RA, and 2 PSS) had between 140
and 400 units. The significance of these low-level
elevations is unclear. The distribution among patient
groups is very different from that of patients with
higher elevations, showing no predilection for DM but
possibly being more frequent in SLE. Most of these
patients had the same low-level elevations on other
ELISA binding tests, while most of those with higher
elevations had clearly negative results on other tests.
Some had detectable serum binding without antigen,
but usually not enough to bring the activity to normal
range when subtracted.
While most of the samples were significantly
inhibited by purified antigen (most losing more than
40% OD), average inhibition of myositis and non-
myalsitis patients who had <400 units (54% and 53%,
respectively) was less than that of the patients with
>400 units (79%). Whole CTE also inhibited nearly all
the sera with low-level binding. However, after CTE
was applied to the anti-Mi immunoadsorbent, and
could no longer inhibit diluted high-level-binding antiMi-positive sera, it was still able to inhibit most lowlevel-binding sera. Removing Mi antigen (by the immunoadsorbent) left the CTE 68% less effective at
inhibiting high-level sera, while only 21% less effective
at inhibiting low-level sera and 14% for normal sera.
The inhibition study findings on 1 patient with 520
ELISA units suggested nonspecific binding (25% decrease in inhibiting effectiveness), and those on 1
patient with 320 ELISA units were consistent with
specific antibody (56% decrease).
Overall, the incidence of definitely positive
ELISA for anti-Mi-2 in myositis patients was 9.4%.
The proportion of adult DM patients who had 2400
units of anti-Mi-2 was 22.2%, and in childhood DM
patients it was 14.3%. Thus, 21.2% of the total DM
population had 2400 units of anti-Mi-2; this is the
same percentage as that which appeared to have true
antibody by antigen inhibition testing. Of patients with
pure PM, 3.4% had 2400 units, although results of
inhibition studies were equivocal on those positive
samples. Of the overall group of PM patients, 2.3%
had elevated binding. The difference in prevalence of
elevated anti-Mi-2 binding between the DM and the
PM groups and between adult patients with pure PM
and those with DM was significant ( P < 0.01 by chisquare with Yates’ correction). The difference in prevalence of precipitating anti-Mi-2 between these groups
was also significant ( P < 0.01).
Of 15 sera tested by the original complement
fixation inhibition (CFI) method (ability of papaindigested patient serum to inhibit Mi serum-CTE complernent fixation), all 6 CFI-negative sera were
ELISA-negative, whereas 4 of 9 CFI-positive sera
werle ELISA-positive (4 of 6 DM, 0 of 3 PM).
This study describes the Mi-2 antigen and Mi-2
antibody as detected by immunodiffusion and ELISA.
Consistent with other myositis-associated antibodies,
anti-Mi-2 is not common. Present in 9.4% of patients,
it is less common than anti-Jo-1 but about as common
as PM-Scl in the overall myositis population (2), and is
the most common specificity in DM. Mi-2 antibodies
have previously been found-by ID only rarely (approx-
80 1
imately 2% of patients) (2,3). The ELISA was useful in
detecting additional patients with lower levels of antibody, because of its greater sensitivity, although ID
with a highly concentrated, partially purified antigen
revealed precipitins in most of them. It had been
hoped that the ELISA would also be helpful by
detecting nonprecipitating antibodies missed by ID,
but in only 1 serum were the findings suggestive of
this. The significance of the low-level elevations is not
clear, but they may not represent anti-Mi-2.
In addition to being immunologically distinct
from Mi-1 Ag by ID, Mi-2 Ag has a number of other
differences from Mi-1. First, we have demonstrated
that Mi serum can fix complement with purified Mi-2
Ag, while the same patient’s serum was unable to fix
complement with purified Mi-1 Ag (6). Second, Mi-2
binds to DEAE at pH 7.0 and requires 0.2M NaCl for
complete elution, whereas Mi-1 does not bind to
DEAE in 0.01M phosphate buffer alone (6). The
apparent molecular weight and activity on PAGE of
Mi-2 is distinct (Mi-1 having a molecular weight of
150,000).Mi-1 is immunologically identical to BGG by
ID (and may in fact be BGG), and anti-Mi-1 reacts
with BGG (7). This resulted in the persistent contamination of the Mi-2 affinity eluate with BGG. Further
steps were required to remove BGG and prevent
confusion of anti-BGG with anti-Mi-2 in the ELISA,
including the addition of BGG to serum as a blocker.
Most of the myositis patients with anti-Mi-2
had no evidence of anti-Mi-1 by ID or by ELISA using
BGG as antigen. While many of the patients who
clearly had anti-Mi-1 by ID (described elsewhere [7])
had low-level elevations on ELISA for anti-Mi-2,
none were definitely positive except patient Mi. Most
of the patients with anti-Mi-1 had SLE. Thus, the 2
systems appear to be independent.
Mi-2 antibodies were highly correlated with
DM. None of the other specificities is primarily associated with DM. Anti-PM-Scl is seen with about equal
frequency in PM and DM, although it is particularly
associated with myositis-scleroderma overlap (2).
Anti-Jo-1 is much more frequently seen in PM than in
DM (3). The other antibodies are most frequently seen
in overlap syndromes (1,4). No features (other than
the presence or absence of the rash) distinguished the
group of patients who had the antibody. Although 3
patients had malignancies, it does not appear that antiMi-2 is a marker for malignancy in DM. The prototype
patient has never developed any recognized malignancy after 10 years. Relatively few children were studied, but anti-Mi-2 is apparently not seen exclusively in
adults. We saw no obvious fluctuation of titers or
ELISA activity with disease activity, but a systematic
study relating ELISA titer to clinical activity has not
been done.
We have not investigated patients with primary
dermatologic conditions, but they do not characteristically have antinuclear antibodies, and anti-Mi-2, if
present, would be seen on routine IIF. We did investigate SLE patients, whose rash histologically resembles DM (14). Thus, it is unlikely that these antibodies
are secondary to the rash. The pathogenesis of the
muscle disease does not seem to be humorally mediated (8), but the clinical association of these antibodies
raised the question of a relationship to the pathogenesis of the rash. Although the antibody is not seen in the
majority of patients with DM and the rash can occur in
agammaglobulinemia patients (14), almost every patient with the antibody has the rash. However, it is
more likely that the clinical associations of the antibody are due to genetic predispositions (for example,
anti-Jo-1 is associated with HLA-DR3 [15]) or to
differences in inciting events (such as different viruses).
It has recently been shown that the Jo-1 antigen
is the enzyme histidyl-tRNA synthetase (16) and that
there may be other autoantibodies in myositis patients
directed at the animoacyl-tRNA synthetase group of
enzymes (17,18). These enzymes are found in the cell
cytoplasm, and one report describes a cytoplasmic
pattern on IIF with Jo antibody (19). It is unlikely that
the nuclear Mi-2 Ag is an aminoacyl-tRNA synthetase.
None of the anti-Mi sera exhibit cytoplasmic staining
on IIF. Possible functional roles for the Mi-2 antigen
are being actively investigated.
The purity of the antigen in our study, as
measured by specific activity, may well be underestimated. The elution from the affinity column involves
harsh conditions that appeared to idactivate a considerable amount of antigen. Complement-fixing activity
was more labile than binding activity and, in some
runs, was completely destroyed. Thus, the extent of
purification measured by complement-fixing activity
was less than that measured by the direct binding
ELISA, and ELlSA activity recovered was less than
expected. To date, attempts at further purification of
the antigen have resulted in loss or inactivation of the
material, but this effort is continuing. We could not
confirm by the Western blot method that either of the 2
bands seen on PAGE was antigenic. Either antigenic
activity was lost during the procedure or during the
separation of the bands, or these bands do not repre-
sent the true Mi-2 antigen. The latter is unlikely
considering the consistency of the bands and the fact
that no other definite bands are seen. Gel filtration of
purified material (not shown) was also unsuccessful at
eluting an antigenic peak to determine its molecular
There is strong correlation between the presence of anti-Mi-2 precipitin and elevation of ELISA
binding. Thus, it is very likely that the ELISA is
measuring the same antibody as the precipitin. There
is also good reason to believe that the ELISA and ID
antibody, Mi-2, is the original CFI antibody. The CFI
results were similar to the ELISA results in most
patients. Although the total percentages of positive
sera in the DM and the PM groups were similar to each
other in the original study, the DM sera were much
better inhibitors of the CF reaction than were the PM
sera (5). Generally, antibodies that fix complement
will also precipitate, and there is direct evidence that
the only other precipitin in Mi serum, anti-Mi-I, does
not fix complement (6) and is not in any way related to
the CF antibody (7).
However, the frequency of the CF-reactive
antibody was much higher than the observed frequency of anti-Mi-2. This may simply represent nonspecific interference in the original study. One possibility is
that the antigen is altered in some way or that a
complex is dissociated by the purification process,
thus losing antigenic epitopes. Alternatively, low affinity antibodies which may be missed by ELISA (20)
may be measured in the CFI assay. Thus, the true
frequency of anti-Mi-2, particularly in DM, may be
higher than indicated by the ELISA data presented
The data presented describe the first serologic
reaction with specificity for dermatomyositis. Continued study of this reaction may improve the sensitivity
of the assay for detection of antibody and may provide
clues to the etiology and pathogenesis of this disease.
The authors wish to thank Dr. Frank Arnett, Dr.
Thomas Bunch, Dr. Paul Goldfarb, and Dr. Sarah Newel1 for
providing sera from patients with myositis, Marcia Anderson
for excellent technical assistance, and Bonnie Wilds for
assistance in the preparation of the manuscript.
1. Reichlin M, Arnett FC Jr: Multiplicity of antibodies in
myositis sera. Arthritis Rheum 27: 1150-1 156, 1984
2. Reichlin M, Maddison PJ, Targoff I, Bunch T, Arnett F,
Sharp G, Treadwell E, Tan EM: Antibodies to a nuclear1
riucleolar antigen in patients with polymyositis overlap
syndromes. J Clin Immunol 4:40-44, 1984
3. Nishikai M, Reichlin M: Heterogeneity of precipitating
amtibodies in polymyositis and dermatomyositis: characterization of the Jo-1 antibody system. Arthritis Rheum
2:3:881-888, 1980
4. Fdimori T, Akizuki M, Yamagata H, Inada S, Yoshida S,
Homma M: Characterization of a high molecular weight
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antibodies, associations, dermatomyositis
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